Protein kinase C (PKC) isoforms are serine threonine kinases that are activated upon agonist stimulation and regulate several functions. In the previous grant period, we demonstrated the functional role of nPKC isoforms in platelets and the molecular interactions of PKC? with SHIP1 and Lyn to regulate differential granule release. We have strong preliminary data indicating that PKC? is phosphorylated on Y155 and Y311, but these two tyrosines are differentially phosphorylated upon stimulation of Collagen or Thrombin receptors. We have generated knock-in mice for PKC? Y155F mutation and our preliminary data point to the regulation of GPVI-mediated functional events by PKC? through the Y155 residue. We also have strong preliminary data suggesting that Y311 might regulate cPLA2 activation and thromboxane generation. Finally, we have identified a negative regulatory role for PKC? in megakaryopoiesis and platelet production. Hence we will focus our efforts on the molecular mechanisms of regulation of platelet function and megakaryopoiesis by PKC? in this grant period. Our overall hypothesis is that PKC? plays a crucial role in platelet function and megakaryopoiesis through tyrosine phosphorylation of Y155 and Y311 residues. We propose that these phosphorylated tyrosines form nodal points for signal transduction through interaction with other signaling molecules thus regulating functional responses. 1) We hypothesize that diverse signaling pathways downstream of G protein-coupled receptors and tyrosine kinase-linked receptors phosphorylate different tyrosine residues on PKC? and these differential phosphorylations modify the functional role of this isoform. We hypothesize that PKC? Y155 phosphorylation regulates dense granule release through association with SHIP1 and Lyn, whereas PKC? Y311 phosphorylation regulates cPLA2 activation and thromboxane generation. We will investigate the functional role of phosphorylation of Y155 and Y311 on PKC? in platelets using PKC? knock-in mice with the specific mutation Y155F or Y311F. We hypothesize that phosphorylated Y155 and Y311 on PKC? initiate different signaling pathways than other tyrosine residues on PKC? through association of different signaling molecules and we will test this hypothesis by biochemical/proteomic approaches. 2) We hypothesize that PKC? plays a distinct role in megakaryocyte differentiation and platelet production. We propose to test this hypothesis using bone marrow megakaryocytes from mice lacking PKC? as well as from Y155F and Y311F knock-in mice. The studies proposed in this application will enhance our understanding of PKC? signaling networks and their regulation of functional responses in platelets and the production of platelets. These studies will then help us identify targets to trea thrombotic disorders as well as thrombocytopenia.